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Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production

Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK

Peter Kelly

Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK

Patrick Vallance

Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK

James Leiper

Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK

Abstract

GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH₄), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH₄ pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH₄ through its protein–protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH₄ nor NO levels but significantly attenuated the rise in BH₄ and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH₄ and NO levels in vivo.

Key Words: cytokines • endothelial cells • lipopolysaccharide • nitric oxide

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